Rapid Detection of bla(KPC) in Carbapenem-Resistant Enterobacterales Based on CRISPR/Cas13a

Klebsiella pneumoniae carbapenemase (KPC) is a crucial enzyme that causes carbapenem resistance in Enterobacterales, and infections by these "superbugs" are extremely challenging to treat. Therefore, there is a pressing need for a rapid and accurate KPC detection test to control the prevalence of carbapenem-resistant Enterobacterales (CREs). In this study, we established a novel method for detection of bla(KPC), the gene responsible for encoding KPC, based on a recombinase polymerase amplification (RPA) and a CRISPR/Cas13a reaction coupled to fluorophore activation (termed RPA-Cas13a assay). We carefully selected a pair of optimal amplification primers for bla(KPC) and achieved a lower limit of detection of approximately 2.5 copies/μL by repeatedly amplifying a recombinant plasmid containing bla(KPC). The RPA-Cas13a assay demonstrated a sensitivity of 96.5% and specificity of 100% when tested on 57 bla(KPC)-positive CRE strains, which were confirmed by DNA sequencing. Moreover, in 311 sputum samples, the theoretical antibiotic resistance characteristics of bla(KPC)-positive strains obtained by the RPA-Cas13a assay were highly consistent with the results of antibiotic susceptibility test (Kappa = 0.978 > 0.81, P < 0.01). In conclusion, the RPA-Cas13a system is a simple and one-hour efficient technology for the detection of a potentially fatal antibiotic resistance gene.